TY - JOUR
T1 - Design, Synthesis, and Biophysical Evaluation of Mechanism-Based Probes for Condensation Domains of Nonribosomal Peptide Synthetases
AU - Shi, Ce
AU - Miller, Bradley R.
AU - Alexander, Evan M.
AU - Gulick, Andrew M.
AU - Aldrich, Courtney C.
PY - 2020/7/17
Y1 - 2020/7/17
N2 - Nonribosomal peptide synthetases (NRPSs) are remarkable modular enzymes that synthesize peptide natural products. The condensation (C) domain catalyzes the key amide bond-forming reaction, but structural characterization with bound donor and acceptor substrates has proven elusive. We describe the chemoenzymatic synthesis of condensation domain probes C1 and C2 designed to cross-link the donor and acceptor substrates within the condensation domain active site. These pantetheine probes contain nonhydrolyzable ketone and α,α-difluoroketone isosteres of the native thioester linkage. Using the bimodular NRPS responsible for synthesis of the siderophore enterobactin as a model system, probe C2 was shown by surface plasmon resonance (SPR) to stabilize an intermolecular interaction between the peptidyl carrier protein (PCP) and C domains in EntB and EntF, respectively, with a dissociation constant of 1-2 nM, whereas the unmodified holo-EntB showed no interaction with EntF. The described condensation domain chemical probes provide powerful tools to study dynamic multifunctional NRPS systems.
AB - Nonribosomal peptide synthetases (NRPSs) are remarkable modular enzymes that synthesize peptide natural products. The condensation (C) domain catalyzes the key amide bond-forming reaction, but structural characterization with bound donor and acceptor substrates has proven elusive. We describe the chemoenzymatic synthesis of condensation domain probes C1 and C2 designed to cross-link the donor and acceptor substrates within the condensation domain active site. These pantetheine probes contain nonhydrolyzable ketone and α,α-difluoroketone isosteres of the native thioester linkage. Using the bimodular NRPS responsible for synthesis of the siderophore enterobactin as a model system, probe C2 was shown by surface plasmon resonance (SPR) to stabilize an intermolecular interaction between the peptidyl carrier protein (PCP) and C domains in EntB and EntF, respectively, with a dissociation constant of 1-2 nM, whereas the unmodified holo-EntB showed no interaction with EntF. The described condensation domain chemical probes provide powerful tools to study dynamic multifunctional NRPS systems.
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U2 - 10.1021/acschembio.0c00411
DO - 10.1021/acschembio.0c00411
M3 - Article
C2 - 32568518
AN - SCOPUS:85088240763
VL - 15
SP - 1813
EP - 1819
JO - ACS Chemical Biology
JF - ACS Chemical Biology
SN - 1554-8929
IS - 7
ER -